Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-85) contains supplementary material, which is available to authorized users.

Nicole Grabinski, Florian Ewald contributed equally to this work.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

NG, FE, BH, KS, US, BN and MJ designed the study. FE, NG and MJ performed the experiments and interpreted the experimental findings. FE and NG drafted the manuscript. FE, NG and MJ wrote the final version of the manuscript. All authors read and approved the final manuscript.

Abstract

Background

Due to the frequent dysregulation of the PI3K/AKT/mTOR signaling pathway, mTOR represents a suitable therapeutic target in hepatocellular carcinoma (HCC). However, emerging data from clinical trials of HCC patients indicate that mTOR inhibition by RAD001 (Everolimus) alone has only moderate antitumor efficacy which may be due to the feedback activation of AKT after mTOR inhibition. In this study, we analyzed the effects of dual inhibition of mTOR and AKT on the proliferation of HCC cell lines. In addition, we measured the feedback activation of each of the AKT isoforms after mTOR inhibition in HCC cell lines and their enzymatic activity in primary samples from HCC patients.

Methods

The activation status of specific AKT isoforms in human HCC samples and corresponding healthy liver tissue was analyzed using an AKT isoform specific in vitro kinase assay. AKT isoform activation after mTOR inhibition was analyzed in three HCC cell lines (Hep3B, HepG2 and Huh7), and the impact of AKT signaling on proliferation after mTOR inhibition was investigated using the novel AKT inhibitor MK-2206 and AKT isoform specific knockdown cells.

Results

AKT isoforms become differentially activated during feedback activation following RAD001 treatment. The combination of mTOR inhibition and AKT isoform knockdown showed only a weak synergistic effect on proliferation of HCC cell lines. However, the combinatorial treatment with RAD001 and the pan AKT inhibitor MK-2206 resulted in a strong synergism, both in vitro and in vivo. Moreover, by analyzing primary HCC tissue samples we were able to demonstrate that a hotspot mutation (H1047R) of PI3KCA, the gene encoding the catalytic subunit of PI3K, was associated with increased in vitro kinase activity of all AKT isoforms in comparison to healthy liver tissue of the patient.

Conclusion

Our results demonstrate that dual targeting of mTOR and AKT by use of RAD001 and the pan AKT inhibitor MK-2206 does effectively inhibit proliferation of HCC cell lines. These data suggest that combined treatment with RAD001 and MK-2206 may be a promising therapy approach in the treatment of hepatocellular carcinoma.

Anzeige

Zusatzmaterial

Additional file 1: Figure S1. No increase in AKT phosphorylated at T308 or S473 is detectable in Hep3B cells after RAD001 treatment. Hep3B cells were treated with 100 nM RAD001 up to 72 h, and cell lysates were prepared at the indicated time points. Where indicated, medium was removed after 48 h and replaced by fresh, 100 nM RAD001 containing medium. Cell lysates were analyzed for AKT and mTOR signaling. HSC70 was used as loading control. (PDF 62 KB)

Additional file 2: Figure S2. Influence of plating density on proliferation of HCC cell lines. Increasing numbers of HCC cells were seeded into 96-wells and incubated with different concentrations of RAD001 for 72 h. Proliferation was subsequently analyzed by BrdU incorporation. One representative experiment out of two is shown. (PDF 203 KB)

Additional file 6: Figure S6. Treatment of mice bearing subcutaneous HCC-tumors had no effect on body weight. Mice treated with Placebo, RAD001, MK-2206 or both compounds in combination were weighed every other day during the first 18 day treatment period. Until day 15, no statistically significant changes in body weight were detected. Weight loss at day 18 in MK-2206 and Placebo treated animals was due to tumor cachexia, and these animals had to be withdrawn from the experiment. Data are presented as mean ± SEM. (PDF 89 KB)